The goal of this laboratory is to develop methods for stable introduction and expression of genes in miniature swine keratinoyctes for gene therapy purposes. The in vivo approach directly introduces the gene into epidermis while in our ex vivo approach, we first isolate keratinocytes, insert the desired gene while in tissue culture, and graft the genetically modified keratinoyctes back onto the donor. We have developed a novel in vivo approach for directly introducing and expressing genes in epidermis by injecting naked plasmid DNA into the dermis underlying the epidermis. The naked DNA traverses the dermal-epidermal junction and is absorbed and expressed by keratinocytes throughout the epidermis. Human skin organ cultures are also able to express injected naked DNA in the epidermis. Since skin is a very potent immunologic organ, direct injection of DNA is being used to develop vaccines for Leishmania. Direct injection of DNA expressing Leishmania surface proteins are able to induce a TH1 cell-mediated immune response to Leishmania. Another use of this approach is the expression of biological response modifiers such as cytokines and interferons. In a dog papilloma model, plasmids expressing interferon-alpha can be injected into and expressed in the epidermal keratinocytes of papillomas, leading to regression of the papilloma. The ex vivo approach can ensure that a much higher percentage of the keratinoyctes have the desired gene stably integrated into the genome, ensuring persistent expression. Currently, the ex vivo approach is being combined with topical (in vivo) selection for those keratinocytes containing the gene of interest. Specifically, keratinoyctes containing a selectable marker gene, such as MDR, along with the desired gene, can be selected using topical colchicine or related agents. The MDR gene has been transduced into primary pig keratinoytes using defective retroviral vectors and we have determined the appropriate colchicine formulations and dose/concentrations for in vivo topical selection. Methods for grafting pig keratinocyts (skin equivalents) back onto the donor pigs have been developed. Finally, we have begun to investigate how a POU transcription factor, that is specifically expressed in epidermis, might influence and regulate epidermal differentiation. In vitro models of epidermal differentiation have been developed where keratinocytes are transduced with retroviral vectors containing the POU transcription factor and transgenic models have been made in which the POU trancription factor are either misexpressed in abnormal locations of the epidermis or mutated POU genes are expressed at the appropriate epidermal layers.